CN112574310B - anti-SIRP alpha antibodies and uses thereof - Google Patents

anti-SIRP alpha antibodies and uses thereof Download PDF

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CN112574310B
CN112574310B CN202011464010.2A CN202011464010A CN112574310B CN 112574310 B CN112574310 B CN 112574310B CN 202011464010 A CN202011464010 A CN 202011464010A CN 112574310 B CN112574310 B CN 112574310B
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antibody
antigen
ser
binding fragment
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CN112574310A (en
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王海彬
吴振华
周雅琼
聂磊
陈瑶
蒋美珠
焦静雨
杨亚平
高栋
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Zhejiang Borui Biopharmaceutical Co ltd
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Priority to JP2023558929A priority patent/JP2023553758A/en
Priority to EP21902685.3A priority patent/EP4261223A1/en
Priority to KR1020237021265A priority patent/KR20230113348A/en
Priority to CN202180012639.XA priority patent/CN115052895A/en
Priority to IL303554A priority patent/IL303554A/en
Priority to US18/265,777 priority patent/US20240034804A1/en
Priority to PCT/CN2021/136763 priority patent/WO2022121980A1/en
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/77—Internalization into the cell
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention belongs to the field of biology, and relates to an anti-SIRP alpha antibody or an antigen binding fragment thereof, a composition and application thereof. The SIRPalpha antibody and the antigen binding fragment thereof can effectively block the combination of SIRPalpha and CD47, further effectively promote the phagocytosis of tumor cells by macrophages, effectively inhibit the growth of tumors, and have good patent medicine prospect.

Description

anti-SIRP alpha antibodies and uses thereof
Technical Field
The invention belongs to the field of biology, and relates to an anti-SIRP alpha antibody.
Background
Sirpa is a transmembrane protein expressed on the surface of myeloid cells such as macrophages, monocytes, dendritic cells, granulocytes, etc., and belongs to an immunoglobulin superfamily (IgSF) member. SIRPalpha consists of an N-terminal extracellular region containing three IgSF domains (Ig-V domains at the N-terminal), a transmembrane region, and a C-terminal intracellular region containing a tyrosine immune receptor inhibitory motif (ITIM). The primary ligand of sirpa is CD47, a class of transmembrane glycoproteins that are widely expressed in normal and diseased tissues. Binding of CD47 to sirpa on myeloid cells such as macrophages phosphorylates ITIM in the intracellular region of sirpa, thereby recruiting and activating phosphatases SHP-1 and SHP-2, and thereby inhibiting phagocytic function of macrophages and the like through downstream signaling. Normal tissues release the "do not eat me" signal by expressing CD47 and CD 47-sirpa signaling as a self-protecting mechanism.
It was found that CD47 is overexpressed on almost all tumor cells that use CD47 binding to sirpa to release a "stuttering me" signal to evade immune cell monitoring. And the clinical CD47 expression level is closely related to the poor prognosis of the patient. In vivo and in vitro studies have also found that blocking CD 47/sirpa can promote tumor cell phagocytosis and inhibit tumor growth in animal models. The above shows that targeting CD 47/sirpa can be used as a new way of development of tumor immunotherapy drugs, considering the broad expression of CD47 on normal cell tissues, especially on erythrocytes and platelets, targeting CD47 may bring about a certain blood toxicity, and the proteins interacting with CD47 besides sirpa also include TSP-1, integrins, etc., the signaling pathway involved in CD47 is more complex, and targeting CD47 has more risks, therefore, developing an anti-sirpa antibody to block the CD 47/sirpa signaling pathway may be a more effective strategy for tumor drug development.
Disclosure of Invention
The invention aims to provide a novel SIRPalpha antibody which can be specifically combined with SIPRa and has great potential in tumor treatment.
In a first aspect, the invention provides an anti-sirpa antibody or antigen-binding fragment thereof that binds sirpa or a fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three CDRs, VH CDR1, VH CDR2 and VH CDR3 respectively, and a light chain variable region comprising three CDRs, VL CDR1, VL CDR2 and VL CDR3 respectively; wherein, the liquid crystal display device comprises a liquid crystal display device,
VH CDR1 comprises SEQ ID NO: 3. 13, 23 or 33, or a fragment thereof, VH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4. 14, 24 or 34, or a fragment thereof, VH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5. 15, 25 or 35, or a VL CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8. 18, 28 or 38, or a VL CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9. 19, 29 or 39, or a VL CDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10. 20, 30 or 40 or a fragment thereof.
In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3; wherein, the liquid crystal display device comprises a liquid crystal display device,
the amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 3;
the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 4;
the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 5;
the amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 8;
the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 9;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 10.
In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3; wherein, the liquid crystal display device comprises a liquid crystal display device,
the amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 13;
the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 14;
the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 15;
the amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 18;
the amino acid sequence of VL CDR2 is shown as SEQ ID NO. 19;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 20.
In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3; wherein, the liquid crystal display device comprises a liquid crystal display device,
the amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 23;
the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 24;
the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 25;
the amino acid sequence of VL CDR1 is shown as SEQ ID NO. 28;
the amino acid sequence of VL CDR2 is shown as SEQ ID NO. 29;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 30.
In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3; wherein, the liquid crystal display device comprises a liquid crystal display device,
the amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 33;
the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 34;
the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 35;
the amino acid sequence of VL CDR1 is shown as SEQ ID NO. 38;
the amino acid sequence of VL CDR2 is shown as SEQ ID NO. 39;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 40.
In some embodiments, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO. 1, 11, 21 or 31, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.
In some embodiments, the light chain variable region has an amino acid sequence as set forth in SEQ ID NO. 6, 16, 26 or 36, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.
In certain preferred embodiments, the 3 CDRs contained in the heavy chain variable region, and/or the 3 CDRs contained in the light chain variable region, are defined by the Kabat or Chothia numbering system.
In some embodiments, an antibody or antigen binding fragment thereof of the invention may further comprise one or more of a heavy chain constant region, a light chain constant region, an Fc region. In a further preferred embodiment, the light chain constant region is a lambda chain or a kappa chain constant region. In some preferred embodiments, the antibody or antigen binding fragment thereof is of the IgG1, igG2, igG3 or IgG4 type.
In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.
In a second aspect, there is provided a nucleic acid molecule comprising a nucleotide encoding an antibody or antigen binding fragment thereof of the invention. In some embodiments, the nucleic acid molecule encodes the heavy chain variable region and/or the light chain variable region of the antibody or antigen binding fragment thereof.
In preferred embodiments, the nucleic acid molecule encodes a heavy chain variable region having a nucleotide sequence as set forth in SEQ ID NO. 2, 12, 22 or 32, or having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto. In other preferred embodiments, the nucleic acid molecule encodes a light chain variable region having a nucleotide sequence as set forth in SEQ ID NO. 7, 17, 27 or 37, or having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.
In a third aspect, the present invention provides a biomaterial, comprising:
(1) A vector, host cell, microorganism, or the like comprising the nucleic acid molecule of the invention; or (b)
(2) Expression products, suspensions, supernatants, etc. of the above (1).
The person skilled in the art can easily select and prepare a vector, a host cell or a microorganism comprising the coding sequence of the antibody according to the amino acid sequence of the antibody and know how to culture such a host cell or microorganism, thereby obtaining the corresponding expression product, suspension, supernatant, etc. to obtain the corresponding antibody. This is a conventional technical means in the art.
In a fourth aspect, there is provided a composition comprising an antibody or antigen-binding fragment thereof of the invention; preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.
In a fifth aspect, there is provided a method of making an antibody or antigen-binding fragment thereof of the invention comprising: culturing the above-described host cell to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment.
In a sixth aspect, there is provided the use of an antibody of the invention or an antigen-binding fragment thereof or a nucleic acid molecule of the invention or a biological material of the invention or a composition of the invention in the manufacture of a medicament for the treatment of a tumor; preferably, the tumor is a CD47 expressing positive tumor; further preferably, the tumor is a variety of hematological tumors and solid tumors, such as leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, gastric cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, renal cancer, lung cancer, prostate cancer, ovarian cancer, thyroid cancer, glioma, and the like.
In a seventh aspect, there is provided the use of an antibody of the invention or an antigen-binding fragment thereof or a nucleic acid molecule of the invention or a biological material of the invention or a composition of the invention in the preparation of a formulation that blocks the binding of sirpa and CD 47.
In an eighth aspect, there is provided the use of an antibody of the invention or an antigen-binding fragment thereof or a nucleic acid molecule of the invention or a biological material of the invention or a composition of the invention in combination with one or more other cancer therapeutic agents for the manufacture of a medicament for the treatment of a tumor; preferably, the tumor is a CD47 expressing positive tumor.
In some preferred embodiments, the other cancer therapeutic agents include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, and biomacromolecule drugs. Further preferably, the biomacromolecule drug is a monoclonal antibody drug targeting a tumor cell surface antigen, including an anti-CD 20 antibody (such as ze Bei Tuo mab or rituximab), cetuximab, or trastuzumab.
The SIRPalpha antibody and the antigen binding fragment thereof are blocking antibodies, can effectively block the combination of SIRPalpha and CD47, further effectively promote the phagocytosis of tumor cells by macrophages, effectively inhibit the growth of tumors, and have good patent medicine prospect.
Drawings
Figure 1 shows the blocking effect of murine monoclonal antibodies on sirpa binding to CD 47.
Figure 2 shows the binding of humanized monoclonal antibodies to sirpa.
Figure 3 shows the blocking effect of humanized monoclonal antibodies on sirpa binding to CD 47.
Figure 4 shows the blocking effect of antibody #14 and KWAR23 on sirpa and CD47 binding.
Figure 5 shows the binding kinetics of the #14 antibody to recombinant sirpa protein.
Figure 6 shows the promotion of phagocytosis by antibody #14 against CD 20.
FIG. 7 shows the anti-tumor efficacy of antibody #14 in MC38 tumor models.
Figure 8 shows that antibody #14 and anti-CD 20 synergistically inhibited tumor growth.
Detailed Description
The present invention will be described below with reference to specific examples. Reagents and apparatus used in the following methods are all those commonly used in the art and are commercially available, unless explicitly stated otherwise; the methods used are all conventional in the art and can be carried out unambiguously by a person skilled in the art on the basis of the description of the examples and with corresponding results.
Definition:
in the present invention, the term "antibody" is an immunoglobulin capable of specifically recognizing and binding an antigen, which encompasses a variety of antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, or antibody fragments.
The term "variable region" refers to the domain of an antibody heavy or light chain that recognizes and specifically binds an epitope.
CDR regions or "complementarity determining regions" refer to regions of an antibody variable region that are hypervariable in sequence and form structurally defined loops and/or contain antigen-contacting amino acid residues. The CDRs are mainly responsible for the binding of antibodies to epitopes, determining the specificity of antibodies. In a given heavy or light chain variable region amino acid sequence, the specific amino acid sequence of each CDR is determined using any one or a combination of a number of well-known numbering rules including, for example, kabat, contact, abM and Chothia. The CDRs of the antibodies of the invention can be determined according to any rule in the art or combination thereof.
The invention of the SIRP alpha antibody
The present invention provides anti-sirpa antibodies with high affinity for human sirpa proteins. The antibody can effectively inhibit the combination of SIRPalpha and a ligand CD47 thereof, thereby blocking the downstream signal transmission of CD 47/SIRPalpha, promoting the phagocytosis of macrophages on tumor cells and further realizing the clearance of the tumor cells. The anti-sirpa antibodies described herein, once bound to sirpa proteins on the cell surface, can cause endocytosis, resulting in a decrease in the amount of expression of sirpa proteins on the cell surface, thereby further reducing CD47 signaling with sirpa.
The anti-sirpa antibodies or antigen-binding fragments thereof of the invention comprise substitutions, insertions, or deletions. The SIRPalpha antibodies of the present invention include modifications to the light chain variable region, heavy chain variable region, light chain or heavy chain that differ in amino acid sequence from the amino acid sequence from which the antibody was derived. For example, an amino acid sequence derived from the same designated protein may be similar to the starting sequence, e.g., have a certain percentage identity, e.g., it may be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% identical to the starting sequence.
In the present invention, "identity" refers to the percentage of bases (or amino acids) in two sequences that are compared when the sequences are aligned between two peptides or between two nucleic acid molecules. The alignment and percent homology or sequence identity may be determined using software programs known in the art, such as those described in Ausubel et al eds. (2007) in Current Protocols in Molecular Biology. Preferably, the alignment is performed using default parameters. One such alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP.
In certain embodiments, amino acid modifications, which may be one or more, may be introduced into the Fc region of an antibody provided herein, thereby producing an Fc variant. An Fc variant may comprise a human Fc region sequence comprising amino acid modifications at one or more amino acid positions.
"antibodies and antigen binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, deimmunized antibodies, or Fab fragments, fab 'fragments, F (ab') 2 fragments, single chain antibodies, nanobodies, and epitope-binding fragments of any of the foregoing.
In some embodiments, the antibodies of the invention may be monospecific, bispecific, or multispecific. An anti-sirpa antibody may be linked to another antibody or antibody fragment to produce a bispecific or multispecific antibody with a second or more binding specificities.
In certain embodiments, the antibodies may be further modified to add functional components, moieties suitable for antibody derivatization include, but are not limited to, PEG, dextran, proteins, lipids, therapeutic agents, or toxins. Antibodies may be modified by phosphorylation, acetylation, glycosylation, pegylation, amidation, or ligation with other proteins, and the like.
Tumor treatment methods and antibody uses
The anti-SIRPalpha antibodies, antigen-binding fragments thereof and pharmaceutical compositions comprising the same provided by the invention can be used for diagnosis, prognosis, treatment or inhibition of cancer. The present invention relates to methods of treating cancer in a subject by administering an anti-sirpa antibody or fragment thereof of the invention to a subject in need thereof. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including variants and derivatives of the invention) and nucleic acids or polynucleotides encoding antibodies of the invention (including variants and derivatives of the invention).
The invention also provides combination therapies comprising the anti-SIRPalpha antibodies of the invention in combination with at least one additional therapeutic agent, including but not limited to chemotherapeutic agents, radiotherapeutic agents and bio-macromolecular drugs. In one embodiment, the biomacromolecule drug is a monoclonal antibody drug targeting a tumor cell surface antigen, including anti-CD 20 antibodies (e.g., ze Bei Tuoshan antibody, rituximab), cetuximab, and trastuzumab.
The antibodies of the invention (and any additional therapeutic agents) may be administered by any suitable means, including, but not limited to, intraperitoneal, intravenous, subcutaneous, intranasal, intramuscular injection. The antibodies, and variants or compositions thereof, may be administered by any convenient route, for example by bolus injection or infusion, by absorption through the epithelium or skin mucosa.
The invention of the example of the SIRP alpha antibody sequence
Table 1: amino acid sequence numbering of VH, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, VL-CDR3 of an antibody of the invention
Figure GDA0003889853690000071
Table 2: VH, VL DNA sequence numbering of antibodies of the invention
Antibody numbering VH DNA sequence numbering VL DNA sequence numbering
#
4 2 7
#11 12 17
#13 22 27
#14 32 37
Table 3: sequence information of the present invention
Figure GDA0003889853690000081
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Figure GDA0003889853690000091
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Figure GDA0003889853690000101
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Figure GDA0003889853690000111
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Figure GDA0003889853690000121
TABLE 4#14 heavy and light chain amino acid sequences of antibodies
Figure GDA0003889853690000122
Example 1: preparation of SIRP alpha antibody mouse monoclonal antibody
This example describes a method for preparing a mouse anti-human sirpa monoclonal antibody using hybridoma technology. The extracellular region SIRPalpha protein (Uniprot: P78324) is expressed as an immunogen, an Fc tag is added at the C terminal of the extracellular region SIRPalpha protein amino acid sequence (Glu 31-Arg 370), and then cloned on a V152 vector (supplied by Hua' an monoclonal antibody) to obtain V152-SIRPalpha ECD-Fc. 293 cells (origin: ATCC) were transiently transfected, and after 5 days the cell culture broth was collected and the supernatant was purified with protein A (manufacturer: solarbio, cat# I8090). To prepare an anti-human SIRP alpha mouse monoclonal antibody, 100 μg SIRP alpha egg is first usedBAL B/c mice (supplied by wakamizumab) were immunized on white 4 weeks old. The immunized mice were re-immunized with 50 μg sirpa protein on days 14 and 28 after the first immunization. ELISA was used to measure serum titers of immunized mice, SIRPalpha was diluted to 1. Mu.g/ml in PBS (manufacturer: china fir gold bridge, cat. No. ZLI-9062), and the microplates were coated overnight at 4 ℃. Blocking with 1% BSA-PBS blocking solution at 37deg.C for 1 hr; after washing the plates with PBST, serum dilutions from immunized mice were added to the plates and incubated for 1 hour at 37 ℃. HRP-labeled goat anti-mouse IgG (manufacturer: abcam, cat# Ab205719,1:10000 dilution) was added to the wash plate and reacted at 37℃for 0.5 hours. Washing the plate, adding TMB solution (manufacturer: huzhou Yingchuang, product number: TMB-S-00), reacting at room temperature for 5 min in the absence of light, and then adding 2N H 2 SO 4 The reaction was terminated. The absorbance was measured at a wavelength of 450nm on a microplate reader. On day 42 post immunization, mice with sufficient titers of anti-sirpa antibodies were boosted with 50 μg sirpa protein. Mice were sacrificed 3-5 days later, spleen cells were collected, and cells were washed 2-3 times by centrifugation with IMDM (manufacturer: shanghai source culture, cat# L610 KJ) basal medium, then mixed with mouse myeloma cells SP2/0 (supplied by Hua' an monoclonal antibody) at a ratio of 1:1, PEG (manufacturer: roche, cat# 25771700) was added to the mixed cells and gently stirred, and left to stand at 37℃for 30s. Diluting the fused cells into IMEM selective medium containing 15% fetal bovine serum (manufacturer: zhejiang Tianzhong organism, product No. 11011-8611) and 1 XHAT (manufacturer: sigma, product No. H0262-1 VL), adding 200 microliters of each well into 96-well cell culture plate, adding 5% CO 2 And placing the mixture in an incubator at 37 ℃. The anti-SIRP alpha antibodies in hybridoma supernatants were detected 10-14 days later by ELISA experiments. 11 different hybridoma clones were identified, including 10F11-5-6, 1B6-1-1, 27A11-1-8, 14B11-5-4, 31D4-4-5, 7C2-3-8, 30C1-6-4, 4A3-5-1, 6H1-8-6, 9A12-5 and 10F4-15, and used for further analysis.
Example 2: binding of murine antibodies to SIRPalpha
ELISA was used to test the binding capacity of murine antibodies to SIRPalpha protein, and the specific method was as described in example 1, wherein the initial murine antibody concentration was 10 μg/ml, and a total of 7 gradients were obtained by 3-fold gradient dilution with PBS. The results are shown in Table 5.
Table 5: EC50 value of binding Activity of murine monoclonal antibody and SIRPalpha
Figure GDA0003889853690000131
Figure GDA0003889853690000141
The binding of murine monoclonal antibodies to SIRP on membrane of human myeloid leukemia mononuclear cells (THP-1, source: china academy of sciences cell bank) endogenously expressing SIRP was analyzed by flow cytometry (FACS). THP-1 cells were reacted with different concentrations of murine mab (highest concentration 1. Mu.g/ml, 3-fold dilution, total of 7 concentration points) at 4℃for 30min. After washing the cells 2 times, FITC-labeled goat anti-mouse IgG (manufacturer: jackson, cat. No. 115-095-003,1:1000 dilution) was added and reacted at 4℃for 30 minutes in the absence of light. After washing the cells 2 times, they were examined with a flow cytometer BD C6. The results are shown in Table 6.
Table 6: EC50 value of murine monoclonal antibody binding to THP-1 cells
Antibody numbering Cloning FACS EC50(pM)
mAb#4 10F11-5-6 139.22
mAb#6 1B6-1-1 231.81
mAb#11 27A11-1-8 125.84
mAb#12 14B11-5-4 211.43
mAb#13 31D4-4-5 144.27
mAb#14 7C2-3-8 128.72
mAb#15 30C1-6-4 128.70
mAb#16 4A3-5-1 232.4
mAb#17 6H1-8-6 544.21
mAb#18 9A12-5 212.34
mAb#19 10F4-15 220.11
Example 3: blocking of SIRPalpha binding to CD47 by murine anti-SIRPalpha antibodies
According to the results of example 2, combining the binding capacity of murine mAb to sirpa and to THP-1 cells, mAb #4, mAb #11, mAb #13 and mAb #14 were the most active molecules, so selection of mAb #4, mAb #11, mAb #13 and mAb #14 examined whether these murine mAb could block sirpa binding to CD 47. CD47-Fc (manufacturer: acrobiosystems, cat. No. CD 7-H5256) was coated on a 96-well ELISA plate and incubated overnight at 4 ℃; after washing the plate, 1% BSA-PBST blocking solution was added and blocked at 37℃for 1 hour. Antibodies (1.8 nM starting, 3-fold dilution, 7 total) and biotin-labeled SIRPalpha (manufacturer: acrobiosystems, cat. No. CDA-H82F 2) were added to the plates and incubated for 1 hour at 37 ℃. After washing the plates, HRP-labeled streptavidin (manufacturer: abcam, cat# ab 7403) was added and reacted at 37℃for 0.5 hours. After washing the plate, TMB solution is added, the reaction is carried out for 30 minutes at room temperature and in the dark, and 2N H is added 2 The reaction was stopped by SO4 and absorbance at 450nm was measured by a microplate reader. IgG (gold synthesis) was used as experimental control. The results are shown in FIG. 1, which shows that these murine antibodies all inhibit SIRPalpha binding to CD47 to varying degrees.
Example 4: humanization of murine antibodies
Based on the results of examples 2 and 3, mAb #4, mAb #11, mAb #13 and mAb #14 were selected for humanization and the heavy and light chain variable regions of these antibodies were compared to a database of available human IgG gene sequences to identify the best matching human germline Ig gene sequences. Specifically, the human germline IgG heavy chains selected by mAb#4, mAb#11, mAb#13 and mAb#14 are IGHV1-69-2 x 01, IGHV2-26 x 01, IGHV7-4-1 x 01, IGHV4-31 x 02, and the human germline IgG light chains selected by mAb#4, mAb#11, mAb#13 and mAb#14 are IGKV4-1 x 01, IGKV1-9 x 01, IGKV4-1 x 01 and IGKV4-1 x 01, respectively. The CDR regions of mab#4, mab#11, mab#13 and mab#14 heavy chains were grafted onto the framework sequences of the matched heavy chain variable region genes, and the CDR regions of mab#4, mab#11, mab#13 and mab#14 light chains were grafted onto the framework sequences of the matched light chain variable region genes. The novel humanized molecules are designated #4, #11, #13 and #14, respectively. After construction, the sequences of the heavy chain variable region, the light chain variable region and the CDR regions of the respective humanized antibodies obtained are shown in table 3. The heavy and light chain sequences of antibody #14 are shown in table 4. The heavy and light chain constant regions of antibodies #4, #11 and #13 are identical to those of antibody #14. The complete amino acid sequences of the heavy and light chains of the antibodies #4, #11 and #13 are fully known to those skilled in the art based on the above disclosure.
The antibodies of the invention were expressed and purified in 293 cells (source: ATCC). The heavy and light chain coding sequences of the antibodies were first cloned into V152 vectors (supplied by Waan monoclonal antibody) and then the V152 vectors with the heavy and light chain coding sequences of the antibody molecules were transferred into 293 cells using the transfection reagent PEI (manufacturer: polyscience, cat# 23966-2). Plasmid DNA and transfection reagent were prepared in a biosafety cabinet, and after mixing the plasmid DNA and PEI (mass ratio: 0.15:1.75) well, the mixture of DNA and transfection reagent was allowed to stand for 10min, gently poured into 293 cells and mixed well. At 37℃with 5% CO 2 After 5 days of culture under the conditions, the cell culture supernatant was centrifuged at 3000rpm for 10min. Collecting supernatant, purifying with protein A (manufacturer: solarbio, cat# I8090) to obtain antibody with purity>95％。
Example 5: binding of humanized antibodies to SIRP alpha proteins
ELISA was used to detect the binding of the humanized antibody to SIRP alpha protein. SIRPalpha (manufacturer: acrobiosystems, cat. No. SIA-H5225) was diluted to 1. Mu.g/ml in PBS and coated on microplates overnight at 4 ℃. Blocking with 1% BSA-PBS blocking solution at 37deg.C for 1 hr; after washing the plates with PBST, humanized antibodies were diluted to different concentrations (1.8 nM starting, 3-fold dilution, 11 total concentrations) and added to the plates and incubated for 1 hour at 37 ℃. Wash plates were loaded with HRP-labeled goat anti-human IgG (manufacturer: abcam, cat. Ab98595,1:10000 dilution) and reacted at 37 ℃ for 0.5 hours. After washing the plate, TMB solution is added, the reaction is carried out for 5 minutes at room temperature and in the dark, and then 2N H is added 2 SO 4 The reaction was terminated. The absorbance was measured at a wavelength of 450nm on a microplate reader. Results are shown in figure 2, EC50 values for sirpa binding for antibodies #4, #11, #13 and #14 are: 1.833nM, 0.4642nM, 0.9517nM and 0.6831nM, which shows that the humanized molecules have higher binding activity with SIRP alpha.
Example 6: blocking of sirpa binding to CD47 by humanized anti-sirpa antibodies
The blocking effect of humanized anti-sirpa antibodies #4, #11, #13 and #14 on sirpa binding to CD47 was tested as described in example 3, and the IC50 of the antibodies #4, #11, #13 and #14, respectively, were shown in fig. 3: 6.03nM, 0.2086nM, 1.5nM and 0.1315nM, show that these humanized antibodies are effective in blocking SIRP alpha binding to CD47, with #11 and #14 having a stronger blocking effect than the other humanized antibodies.
KWAR23 was generated from the sequences disclosed in US2018037652 (SEQ ID NO:1, SEQ ID NO: 2) using KWAR23 as a positive control, and the blocking effect of antibody #14 and KWAR23 on SIRPalpha binding to CD47 was further compared. As a result, as shown in FIG. 4, the IC50 value of the #14 antibody was 3.404nM and the IC50 value of KWAR23 was 13.54nM, indicating that the #14 antibody had a stronger blocking effect on SIRPalpha and CD47 than KWAR23.
Example 7: binding kinetics of antibodies to SIRPalpha
Antibody-to-antigen binding kinetics employs Octet Red96 (manufacturer: forteBio) as the test instrument and an H1S1K biosensor (manufacturer: forteBio) as the test sensor, which can be used to capture antigen directly, followed by immersion of the sensor in the analyte sample (antibody). The experiment comprises five steps: 1. baseline (100 s), 2, loading (capture antigen sirpa) (500 s), 3, baseline (100 s), 4, association (binding antibody) (500 s), 5, association (Dissociation of antibody) (1000 s). After the test was completed, the sensor was regenerated by five cycles of alternating immersion for 5 seconds with regeneration buffer (glycine, pH 1.5) and neutralization buffer (PBS). The running buffer in this experiment was PBS.
Sample treatment: the antigen sirpa protein was diluted to a working concentration of 2.5 μg/mL using running buffer, and analyte samples (# 14 antibody and KWAR 23) were diluted gradient to five working concentrations: 1. Mu.g/ml, 0.5. Mu.g/ml, 0.25. Mu.g/ml, 0.125. Mu.g/ml, 0.0625. Mu.g/ml. Data analysis response signal values (coupled analyte sample signal minus blank analyte sample signal) were calculated using Octet Data Analysis (version 7.0or the test) and the data fitted using a 1:1 binding model.
Binding kinetics fit is shown in FIG. 5 and binding constants, dissociation constants and equilibrium constants are shown in Table 7. The #14 antibody can be seen to have an extremely high affinity, comparable to the positive control antibody KWAR23.
Table 7: binding kinetics of antibody #14 and KWAR23 to SIRPalpha
Sample name Kon Koff KD(M)
#14 1.33×10 6 2.89×10 -5 2.173×10 -11
KWAR23 2.90×10 6 1.48×10 -4 5.094×10 -11
Example 8: the invention provides a method for promoting the effect of SIRP alpha antibody on phagocytic tumor cells of macrophages, which uses PBMC cells (manufacturer: allcels, product number: PB 004F-C) to prepare a basal medium of 1640 (manufacturer: gibco, product number: 22400-089) in 5% CO 2 Culturing at 37 deg.C for 2-3 hr, and gently suckingThe non-adherent cells were removed and induction was initiated by addition of induction medium (80 ng/ml M-CSF). Fresh medium containing sufficient cytokines was changed every 3 days. Macrophages were obtained by culturing until day 7. Raji cells (origin: china academy of sciences cell bank) were fluorescently labeled according to the instructions of CFSE reagent (manufacturer: abcam, cat# AB 113853). The labeled target cells Raji cells and the above-mentioned differentiated macrophages were mixed uniformly at a ratio of 3:1, and an anti-CD 20 antibody (Zuberitamab) was added thereto at a concentration of CAS RN:2251143-19-6,WHO Drug Information,Vol33,No.4,2019,914-915: 0.05 mug/ml, 0.5 mug/ml, manufacturer: marine norboril), or an anti-CD 20 antibody (concentration: 0.05 μg/ml, 0.5 μg/ml) and #14 antibody (concentration: 10 μg/ml), or an anti-CD 20 antibody (concentration: 0.05 μg/ml, 0.5 μg/ml) and KWAR23 antibody (concentration: 10. Mu.g/ml) at 37℃with 5% CO 2 Incubate for 4 hours. After washing the cells with PBS, anti-CD 14-APC (manufacturer: BD Biosciences, cat# 555399) was added and incubated at 4℃for 30min in the absence of light. After washing the cells, the phagocytosis rate was analyzed by flow cytometry and calculated as follows: phagocytosis (%) = (apc+cfse) positive cell proportion/APC positive cell proportion 100%. Figure 6 shows that #14 antibody can enhance phagocytic capacity of anti-CD 20 antibodies to Raji cells.
Example 9: the SIRPalpha antibody induces the endocytosis of SIRPalpha on the cell surface
After THP-1 cells (source: china academy of sciences cell bank) were reacted with 10. Mu.g/ml of antibody at 4℃for 30 minutes, the cells were washed 2 times, and divided into two parts, one part was incubated at 37℃for 4 hours, and one part was further incubated at 4℃as a control cell. After washing the cells 2 times, FITC-labeled goat anti-human IgG (manufacturer: abcam, cat# ab 97224) was added and reacted at 4℃for 30 minutes in the absence of light. Cells were washed and analyzed by flow cytometry, and endocytosis was calculated from the decrease in cell surface fluorescence at 37 ℃ relative to 4 ℃. Endocytosis rate was calculated as follows: endocytosis (%) = (fluorescence intensity at 4 ℃ MFI-37 ℃ MFI)/fluorescence intensity at 4 ℃ MFI 100%.
Table 8 shows that the endocytosis rate of antibody #14 was 52.9% and that of KWAR23 was 34.7%, indicating that antibody #14 was more effective in reducing the expression level of SIRP alpha on the cell surface.
Table 8: endocytosis rate of SIRPalpha on THP-1 cell membrane by #14 and KWAR23
Sample of Endocytosis rate (%)
#14 52.9
KWAR23 34.7
Example 10: the SIRP alpha antibody can inhibit tumor growth
The anti-tumor efficacy of the #14 antibody was evaluated in the hCD47-MC38 mouse colon carcinoma tumor model. hCD47-MC38 cells (source: southern model organism) were inoculated into hSIRP alpha/hCD 47 double-humanized C57BL/6 mice (source: southern model organism). Tumor volume is 150mm 3 On the left and right, mice were given antibody #14 (200 μg/mouse) by intraperitoneal injection, and the control was given twice weekly using an equal amount of PBS solution. The results are shown in FIG. 7, in which antibody #14 continuously inhibited tumor growth compared to the control group.
The anti-tumor efficacy of the combination of antibody #14 and anti-CD 20 (Ze Bei Tuo mab, source: hai Zhengbosi) in a lymphoma model was evaluated. Tail veins of Raji-Luc cells (source: baioxia) were inoculated with B-NDG-hSIRP A mice (source: baioxia), and the average imaging signal intensity reached 1X 10 6 At around p/sec, mice were administered prescribed doses of IgG control (provided by the baisai chart, 200 μg/dose), #14 antibody alone (200 μg/dose), anti-CD 20 antibody alone (2 μg/dose), #14 antibody (200 μg/dose) plus anti-CD 20 antibody (2 μg/dose). As shown in FIG. 8, antibody #14And anti-CD 20 antibodies can exert synergistic anti-tumor effects.
Sequence listing
<110> Zhejiang Borui biopharmaceutical Co., ltd
<120> anti-SIRP alpha antibodies and uses thereof
<130> DSP1F203607ZX
<140> 202011464010.2
<141> 2020-12-11
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<400> 27
gacatcgtga tgacccagtc ccccgactcc ctggccgtgt ccctgggcga gcgcgccacc 60
atcaactgca aggcctccca gtccgtgtcc aacgacgtgg cctggtacca gcagaagccc 120
ggccagcccc ccaagctgct gatctactac gcctccaacc gctgcaccgg cgtgcccgac 180
cgcttctccg gctccggctc cggcaccgac ttcaccctga ccatctcctc cctgcaggcc 240
gaggacgtgg ccgtgtacta ctgccaccag gactacatct ccccctacac cttcggcggc 300
ggcaccaagc tcgagataaa a 321
<210> 28
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 28
Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala
1 5 10
<210> 29
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 29
Tyr Ala Ser Asn Arg Cys Thr
1 5
<210> 30
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 30
His Gln Asp Tyr Ile Ser Pro Tyr Thr
1 5
<210> 31
<211> 118
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 31
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly
20 25 30
Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Asp Gly Ser Arg Tyr Asn Asn Pro Ser Leu
50 55 60
Lys Asn Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Glu Tyr Ala Asn Tyr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 32
<211> 354
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
caggtgcagc tgcaggagag cggaccagga ctggtgaagc ctagccagac actgtctctg 60
acctgcacag tgagcggcgg ctctatctcc agcggctact attggtcttg gatcagacag 120
cacccaggca agggcctgga gtggatcggc tacatctcct atgatggcag caggtataac 180
aatccttccc tgaagaaccg ggtgaccatc tctgtggaca catccaagaa tcagttcagc 240
ctgaagctgt cttccgtgac cgccgctgat acagccgtgt actattgcgc tcgcgaggag 300
tacgccaact atttcgctta ctggggccag ggcaccacag tgaccgtgag ctct 354
<210> 33
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 33
Ser Gly Tyr Tyr Trp Ser
1 5
<210> 34
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 34
Tyr Ile Ser Tyr Asp Gly Ser Arg Tyr Asn Asn Pro Ser Leu Lys Asn
1 5 10 15
<210> 35
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 35
Glu Glu Tyr Ala Asn Tyr Phe Ala Tyr
1 5
<210> 36
<211> 113
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 36
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 37
<211> 339
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
gatatcgtga tgacccagtc tcctgactcc ctggccgtga gcctgggcga gagagctaca 60
atcaactgta agtccagcca gtctctgttc tactcttcca accagaagaa ttttctggcc 120
tggtatcagc agaagcccgg ccagccccct aagctgctga tctactgggc tagcaccaga 180
gagtctggag tgcctgaccg cttcaccgga tccggaagcg gaacagactt caccctgaca 240
atcagctctg tgaaggccga ggatctggcc gtgtactatt gccagcagta ctattcttat 300
ccacccacct tcggccaggg cacaaagctc gagatcaag 339
<210> 38
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 38
Lys Ser Ser Gln Ser Leu Phe Tyr Ser Ser Asn Gln Lys Asn Phe Leu
1 5 10 15
Ala
<210> 39
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 39
Trp Ala Ser Thr Arg Glu Ser
1 5
<210> 40
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 40
Gln Gln Tyr Tyr Ser Tyr Pro Pro Thr
1 5
<210> 41
<211> 448
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 41
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly
20 25 30
Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Asp Gly Ser Arg Tyr Asn Asn Pro Ser Leu
50 55 60
Lys Asn Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Glu Tyr Ala Asn Tyr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 42
<211> 220
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 42
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220

Claims (28)

1. An anti-sirpa antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three CDRs, VH CDR1, VH CDR2, and VH CDR3, respectively, and a light chain variable region comprising three CDRs, VL CDR1, VL CDR2, and VL CDR3, respectively; wherein, the liquid crystal display device comprises a liquid crystal display device,
the amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 33;
the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 34;
the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 35;
the amino acid sequence of VL CDR1 is shown as SEQ ID NO. 38;
the amino acid sequence of VL CDR2 is shown as SEQ ID NO. 39;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 40.
2. The anti-sirpa antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region has an amino acid sequence as set forth in SEQ ID No. 31, or has at least 90% sequence identity thereto; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 36 or has at least 90% sequence identity with the sequence.
3. The anti-sirpa antibody or antigen-binding fragment thereof of claim 2, wherein the amino acid sequence of the heavy chain variable region has at least 95% sequence identity to SEQ ID No. 31 and the amino acid sequence of the light chain variable region has at least 95% sequence identity to SEQ ID No. 36.
4. The anti-sirpa antibody or antigen-binding fragment thereof of claim 3, wherein the amino acid sequence of the heavy chain variable region has at least 96% sequence identity to SEQ ID No. 31 and the amino acid sequence of the light chain variable region has at least 96% sequence identity to SEQ ID No. 36.
5. The anti-sirpa antibody or antigen-binding fragment thereof of claim 4 wherein the amino acid sequence of the heavy chain variable region has at least 97% sequence identity to SEQ ID No. 31 and the amino acid sequence of the light chain variable region has at least 97% sequence identity to SEQ ID No. 36.
6. The anti-sirpa antibody or antigen-binding fragment thereof of claim 5, wherein the amino acid sequence of the heavy chain variable region has at least 98% sequence identity to SEQ ID No. 31 and the amino acid sequence of the light chain variable region has at least 98% sequence identity to SEQ ID No. 36.
7. The anti-sirpa antibody or antigen-binding fragment thereof of claim 6, wherein the amino acid sequence of the heavy chain variable region has at least 99% sequence identity to SEQ ID No. 31 and the amino acid sequence of the light chain variable region has at least 99% sequence identity to SEQ ID No. 36.
8. The anti-sirpa antibody or antigen-binding fragment thereof of any one of claims 1-7, further comprising one or more of a heavy chain constant region, a light chain constant region, an Fc region.
9. The anti-sirpa antibody or antigen-binding fragment thereof of claim 8, wherein the light chain constant region is a lambda chain or a kappa chain constant region.
10. The anti-sirpa antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof is of the IgG1, igG2, igG3, or IgG4 type.
11. The anti-sirpa antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.
12. A nucleic acid molecule comprising a nucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-11.
13. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule encodes a heavy chain variable region and/or a light chain variable region of the antibody or antigen binding fragment thereof.
14. The nucleic acid molecule of claim 13, wherein the nucleic acid molecule encodes a heavy chain variable region having a nucleotide sequence as set forth in SEQ ID No. 32 or having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or the nucleic acid molecule encodes a light chain variable region having a nucleotide sequence as set forth in SEQ ID NO. 37, or having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.
15. The invention provides a biological material, which is:
(1) A vector, host cell or microorganism comprising the nucleic acid molecule of any one of claims 12 to 14; or (b)
(2) The expression product, suspension or supernatant of the above (1).
16. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-11.
17. The composition of claim 16, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
18. A method of preparing the antibody or antigen-binding fragment thereof of any one of claims 1-11, comprising: culturing the host cell of claim 15 to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment.
19. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or a nucleic acid molecule according to claims 12 to 14 or a biomaterial according to claim 15 or a composition according to claim 16 or 17 in the manufacture of a medicament for the treatment of a tumour.
20. The use of claim 19, wherein the tumor is a CD 47-positive tumor, which is a hematological tumor or a solid tumor.
21. The use of claim 20, wherein the tumor is leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, gastric cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, renal cancer, lung cancer, prostate cancer, ovarian cancer, thyroid cancer, or glioma.
22. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or a nucleic acid molecule according to claims 12 to 14 or a biological material according to claim 15 or a composition according to claim 16 or 17 for the preparation of a formulation that blocks the binding of sirpa and CD 47.
23. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or a nucleic acid molecule according to claims 12 to 14 or a biological material according to claim 15 or a composition according to claim 16 or 17 in combination with one or more other cancer therapeutic agents for the manufacture of a medicament for the treatment of a tumor.
24. The use of claim 23, wherein the tumor is a CD47 expression positive tumor.
25. The use of claim 23 or 24, wherein the other cancer therapeutic agents include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents and bio-macromolecular drugs.
26. The use of claim 25, wherein the biomacromolecule drug is a monoclonal antibody drug targeting a tumor cell surface antigen.
27. The use of claim 26, wherein the monoclonal antibody is an anti-CD 20 antibody, cetuximab or trastuzumab.
28. The use of claim 27, wherein the anti-CD 20 antibody is ze Bei Tuo mab or rituximab.
CN202011464010.2A 2020-12-11 2020-12-11 anti-SIRP alpha antibodies and uses thereof Active CN112574310B (en)

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CN202011464010.2A CN112574310B (en) 2020-12-11 2020-12-11 anti-SIRP alpha antibodies and uses thereof
US18/265,777 US20240034804A1 (en) 2020-12-11 2021-12-09 ANTI-SIRPa ANTIBODY AND APPLICATION THEREOF
AU2021398150A AU2021398150A1 (en) 2020-12-11 2021-12-09 ANTI-SIRPα ANTIBODY AND APPLICATION THEREOF
KR1020237021265A KR20230113348A (en) 2020-12-11 2021-12-09 Anti-SIPα Antibodies and Uses Thereof
CN202180012639.XA CN115052895A (en) 2020-12-11 2021-12-09 anti-SIRP alpha antibodies and uses thereof
IL303554A IL303554A (en) 2020-12-11 2021-12-09 Anti-sirpa antibody and application thereof
JP2023558929A JP2023553758A (en) 2020-12-11 2021-12-09 Anti-SIRPα antibody and its application
PCT/CN2021/136763 WO2022121980A1 (en) 2020-12-11 2021-12-09 ANTI-SIRPα ANTIBODY AND APPLICATION THEREOF
EP21902685.3A EP4261223A1 (en) 2020-12-11 2021-12-09 Anti-sirp? antibody and application thereof
TW110146004A TWI805121B (en) 2020-12-11 2021-12-09 AN ANTI-SIRPα ANTIBODY AND AN APPLICATION THEREOF
CA3200487A CA3200487A1 (en) 2020-12-11 2021-12-09 Anti-sirp? antibody and application thereof

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US11572412B2 (en) 2021-06-04 2023-02-07 Boehringer Ingelheim International Gmbh Anti-SIRP-alpha antibodies
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EP3493845A4 (en) * 2016-08-03 2020-04-15 The Board of Trustees of the Leland Stanford Junior University Disrupting fc receptor engagement on macrophages enhances efficacy of anti-sirpalpha antibody therapy
AU2018308364C1 (en) 2017-07-26 2023-02-16 Forty Seven, Inc. Anti-SIRP-alpha antibodies and related methods
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US20210155707A1 (en) * 2018-07-10 2021-05-27 National University Corporation Kobe University ANTI-SIRPalpha ANTIBODY
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